Elevated PTTG and PBF predicts poor patient outcome and modulates DNA damage response genes in thyroid cancer.

The proto-oncogene PTTG and its binding partner PBF have been widely studied in multiple cancer types, particularly thyroid and colorectal, but their combined role in tumourigenesis is uncharacterised. Here, we show for the first time that together PTTG and PBF significantly modulate DNA damage response (DDR) genes, including p53 target genes, required to maintain genomic integrity in thyroid cells. Critically, DDR genes were extensively repressed in primary thyrocytes from a bitransgenic murine model (Bi-Tg) of thyroid-specific PBF and PTTG overexpression. Irradiation exposure to amplify p53 levels further induced significant repression of DDR genes in Bi-Tg thyrocytes (P=2.4 × 10-4) compared with either PBF- (P=1.5 × 10-3) or PTTG-expressing thyrocytes (P=NS). Consistent with this, genetic instability was greatest in Bi-Tg thyrocytes with a mean genetic instability (GI) index of 35.8±2.6%, as well as significant induction of gross chromosomal aberrations in thyroidal TPC-1 cells following overexpression of PBF and PTTG. We extended our findings to human thyroid cancer using TCGA data sets (n=322) and found striking correlations with PBF and PTTG expression in well-characterised DDR gene panel RNA-seq data. In addition, genetic associations and transient transfection identified PBF as a downstream target of the receptor tyrosine kinase-BRAF signalling pathway, emphasising a role for PBF as a novel component in a pathway well described to drive neoplastic growth. We also showed that overall survival (P=1.91 × 10-5) and disease-free survival (P=4.9 × 10-5) was poorer for TCGA patients with elevated tumoural PBF/PTTG expression and mutationally activated BRAF. Together our findings indicate that PBF and PTTG have a critical role in promoting thyroid cancer that is predictive of poorer patient outcome.

Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology.

BACKGROUND: BRAF V600E mutation has been reported to show a high specificity for papillary thyroid carcinoma (PTC). Using this marker to upgrade 'indeterminate' or 'suspicious' thyroid fine needle aspiration (FNA) cytology to 'malignant' could potentially allow one-stage therapeutic total thyroidectomy. METHODS: For a 14-month period, FNA cytology specimens in the Thy3-5 categories, which are the UK equivalents of indeterminate (Thy3a, atypical; Thy3f, follicular), suspicious for malignancy (Thy4) and malignant (Thy5) in the Bethesda System, underwent BRAF mutation testing by melt curve analysis. The results were correlated with histology. RESULTS: We tested 123 cytology specimens of which 12 (9.8%) failed. The BRAF mutation rate in the remainder was 16.2% (18/111), with 93 showing the wild-type. Seventeen mutations were V600E and one was non-V600E. The rate of mutation increased significantly (P < 0.0001 if Thy3a and Thy3f were combined) with the cytology category: 1/42 Thy3a (2.4%), 1/36 Thy3f (2.8%), 4/15 Thy4 (26.7%), 12/18 Thy5 (66.7%). All BRAF mutations correlated with PTC on histology, except for one recurrent PTC without histology. One mutation-positive case with Thy3a cytology showed the target lesion to be a 10-mm follicular adenoma on histology with an immediately adjacent 4-mm micro-PTC, in a patient who did not require total thyroidectomy. CONCLUSION: BRAF mutational analysis by melt curve analysis is feasible in routine thyroid cytology, and in our series had a 100% specificity for PTC in subsequent histology. The application of BRAF analysis could be useful for indeterminate cytology, but we suggest that it would be most appropriate and cost-effective for Thy4/suspicious cases, for which it could enable one-stage therapeutic surgery in the context of multidisciplinary discussion. In contrast, the sensitivity is low and there is no role for avoiding diagnostic thyroid surgery if wild-type BRAF is found.

Metachronous testicular teratoma, testicular seminoma and papillary thyroid carcinoma occurring in a single individual: a report of two unrelated cases.

We describe two unrelated men who both developed teratomas in one testis followed by seminomas in the contralateral testis followed by papillary thyroid carcinomas. Neither man had a family history of cancers. Although random occurrence is possible, genetic predisposition and/or environmental influence would seem a likely explanation for this previously unreported combination of tumours.

The impact of clinical guidelines on surgical management in patients with thyroid cancer.

AIMS: Thyroid cancer is an uncommon but highly curable disease if treated optimally. The aim of this study was to determine whether clinical guidelines introduced locally at the beginning of 1999 were associated with better surgical outcome, using radioiodine uptake as a surrogate measure of completeness of thyroidectomy. MATERIALS AND METHODS: We reviewed the medical records of all patients with thyroid cancer referred to a cancer centre (n=176) 3 years before and 3 years after the introduction of guidelines. The uptake of radioiodine in the thyroid bed after thyroidectomy and before radioiodine ablation was used to assess the completeness of primary surgical treatment. RESULTS: The number of new cases referred to our centre increased from 80 in the 1996-1998 period to 94 during 1999-2001. This was largely because of an excess of papillary thyroid cancers. Documentation in the medical records of the pathological primary tumour size improved from 47.5% to 80.8% following the introduction of guidelines. A significant reduction in radioiodine uptake in the thyroid bed was observed following the introduction of guidelines (5.03% +/- 6.82 (SD) vs 2.75% +/- 5.10 (SD); P=0.005). Linear regression analysis of clinical variables indicated that the year of surgery was the only significant factor influencing radioiodine uptake in the thyroid bed (P=0.014). Twelve hospitals within the Northern Cancer Network carried out thyroid surgery for thyroid cancer in the pre-guideline era compared with seven hospitals in the post-guideline era. Surgeons who were members of the regional multidisciplinary thyroid cancer team operated on 35% of cases in the 1996-1998 period and 56.4% in the 1999-2001 period (P<0.01). CONCLUSIONS: The introduction of clinical guidelines in 1999 was associated with a reduction in the size of thyroid remnant after primary surgical treatment. This was accompanied by fewer hospitals undertaking thyroid surgery and more patients being operated on by surgeons who were members of the thyroid cancer multidisciplinary team.

pNR-2/pS2 immunohistochemical staining in breast cancer: correlation with prognostic factors and endocrine response.

Expression of the oestrogen-regulated pNR-2/pS2 protein has been studied in paraffin sections of a series of 172 primary breast cancers using an immunohistochemical technique. Positive staining of tumour cells was found in 117 tumours (68%): most of these tumours contained only a small proportion of positive cells. pNR-2 immunohistochemical staining correlated positively and significantly with the presence of oestrogen receptor. Mean percentages of pNR-2 positive cells were lower in tumours from postmenopausal women. Smaller, better differentiated tumours were significantly more likely to stain positively for pNR-2. The percentages of pNR-2 positive tumour cells in primary tumours and synchronously excised lymph node metastases were very similar. pNR-2 expression showed an unexpected positive association with lymph node metastasis. We were unable to find any significant association between pNR-2 immunohistochemical staining and either time to relapse or overall survival. There was a significant association between pNR-2 expression in primary tumours and response to endocrine therapy on relapse: positive pNR-2 immunohistochemical staining in primary tumours is predictive of response to hormonal therapy on relapse.

Induction of metallothionein and other mRNA species by carcinogens and tumor promoters in primary human skin fibroblasts.

We used nucleic acid hybridization and cDNA cloning techniques to isolate human sequences that respond to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). These clones were used as probes to examine changes of gene expression that occurred after the proliferation of exponentially growing primary human fibroblasts was arrested. Transcript levels detected by these probes were increased coordinately by treatment of the cells with UV light, mitomycin C, TPA, or the UV light-induced extracellular protein synthesis-inducing factor EPIF (M. Schorpp, U. Mallick, H. J. Rahmsdorf, and P. Herrlich, Cell 37:861-868, 1984). Proteins coded for by these transcripts were characterized by hybrid-promoted translation and by cDNA sequencing. One of the cDNA clones was homologous to the metallothionein IIa gene, and one set of related clones selected RNA for the secreted TPA-inducible protein XHF1 (U. Mallick, H. J. Rahmsdorf, N. Yamamoto, H. Ponta, R.-D. Wegner, and P. Herrlich, Proc. Natl. Acad. Sci. USA 79:7886-7890, 1982).

The mammalian genetic stress response.

A number of carcinogenic and cocarcinogenic agents induce new gene products in mammalian cells including primary human skin fibroblasts. These have been defined by cDNA cloning techniques, by protein resolutions in 2D PAGE and by the detection of new enzymatic functions. The uniform and transient genetic reaction is tentatively called the genetic stress response.

UV-induced extracellular factor from human fibroblasts communicates the UV response to nonirradiated cells.

Ultraviolet light enhances the synthesis of at least eight abundant proteins in human fibroblasts within 2 hr. These proteins are identical with those induced by the tumor promoter TPA. The inducing signal is generated by DNA damage, as these proteins are induced by lower doses of UV in fibroblasts from patients with Cockayne's syndrome or Xeroderma pigmentosum. In the supernatant of UV-treated cells, a heat-labile ammonium sulfate precipitable factor of more than 10 kd (EPIF) was detected which, upon transfer to nonirradiated cells, mimicked UV in the UV-induced synthesis of gene products. The response to UV, TPA, or EPIF was inhibited by fluocinolone acetonide, but not by retinoic acid, protease inhibitors, or superoxide dismutase.

Genetic changes in mammalian cells reminiscent of an SOS response.

Prior to the isolation of mammalian DNA repair genes and identification of their gene products, the comparison between the bacterial SOS response and various similar reactions in mammalian cells remains rather speculative. The increasing number of observed phenomena including enhanced DNA repair, virus induction, induced cellular differentiation, and neoplastic transformation, all following DNA damage or arrest of replication, are, however, suggestive of an SOS-like system of growth control and may form an entry into this fascinating area.

Regulation of MHC class II invariant chain expression: induction of synthesis in human and murine plasmocytoma cells by arresting replication.

The expression of the H2 Ia-associated invariant chain (Ii) has been determined by pulse labeling cells with [35S]methionine and resolving the proteins. Expression is maximal in noncycling peripheral B lymphocytes and is reduced upon maturation of B lymphocytes to plasma cells. B cell-derived cell lines behave correspondingly: IgM+ non-secretor cell lines synthesize Ii while plasmocytoma cells do not. Pre-B cell lines are also negative. In all Ii-negative cell lines, the synthesis of Ii is selectively induced by treating the cells with inhibitors of replication.